Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Receptor-ligand CRISPR-Cas9 activation screen reveals that CD55 interacts with HLA-C∗07:01-VRIG tetramers (A) Schematic of the receptor ligand CRISPR-Cas9 activation screen. K562 cells transduced with a genome-wide activation library were stained with a pool of three HLA tetramers (HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, and HLA-C∗07:01-VRIG), and enriched gRNAs in stained cells were identified using NGS. (B) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and top hits are annotated. (C) K562 cells stably expressing dCas9 and transduced with a gRNA upregulating CD55 or a control guide were stained with the HLA-A, -B, -C, or tetramers as in (A) or with HLA-E∗01:01-VMAP tetramers and analyzed by flow cytometry. (D) In vitro co-immunoprecipitation of recombinant CD55-Fc with HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, HLA-C∗07:01-VRIG, or HLA-E∗01:01-VMAP tetramers. (E) Three different cell lines (HeLa, PC-3M, or SiHa) that express CD55 endogenously were stained for CD55 (top) or with HLA-C∗07:01-VRIG tetramers (bottom) and analyzed by flow cytometry. (F) HeLa wild-type or HeLa CD55 KO cells were stained with αCD55 or HLA-C∗07:01-VRIG tetramers and analyzed by flow cytometry. All data except (B) represent at least three independent experiments. CRISPRa, CRISPR activation screen; TMs, tetramers; WT, wild-type; KO, knockout. Related to Figure S1 and Table S1 .

Article Snippet: Anti-human CD55 (clone BRIC216) , Bio-Rad , Cat: MCA914T; RRID: AB_1102203.

Techniques: CRISPR, Activation Assay, Transduction, Genome Wide, Staining, Stable Transfection, Expressing, Control, Flow Cytometry, In Vitro, Immunoprecipitation, Recombinant, Knock-Out

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Interaction of CD55 with HLA-C∗07:01-VRIG tetramers is allotype and peptide specific (A) HEK293T cells were transfected with a plasmid containing GFP and a truncation mutant of CD55 and analyzed by flow cytometry. GFP+ positive cells were analyzed for staining with HLA-C∗07:01-VRIG. Each mutant removes an additional SCR domain from CD55. Data are represented as mean ± SD. (B) HeLa cells were stained with HLA-C∗07:01-VRIG tetramers after pre-incubation with CD55 blocking antibodies targeting different SCR domains on CD55 and analyzed by flow cytometry. (C) HeLa cells were stained with either HLA-C∗07:01 or HLA-C∗07:02 tetramers loaded with the VRIG peptide and analyzed by flow cytometry. (D) HeLa cells were stained with HLA-C∗07:01 tetramers loaded with different alanine mutants of the VRIGHLYIL peptide and analyzed by flow cytometry. (E) CD55-Fc was immobilized on a Prot-G chip for SPR data using HLA-C∗07:01-VRIG tetramers as analyte to determine interaction on and off rates and K D . Response units were measured with increasing concentrations of HLA-C∗07:01-VRIG tetramers. All data represent at least three independent experiments, except (E), which represents a biological duplicate. FL, full length. Related to Figure S2 , Tables S2 and .

Article Snippet: Anti-human CD55 (clone BRIC216) , Bio-Rad , Cat: MCA914T; RRID: AB_1102203.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Flow Cytometry, Staining, Incubation, Blocking Assay

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet:

Article Snippet: Anti-human CD55 (clone BRIC216) , Bio-Rad , Cat: MCA914T; RRID: AB_1102203.

Techniques: Virus, Recombinant, Blocking Assay, Genome Wide, Activation Assay, CRISPR, Knock-Out, Mutagenesis, Plasmid Preparation, Software, Imaging

Journal: Oncology Letters

Article Title: Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen

doi: 10.3892/ol.2018.8478

Figure Lengend Snippet: Expression of CD55 and CD59 in tumor cells. (A) Cell lines were incubated with fluorescein isothiocyanate-conjugated anti-human CD55 and CD59 monoclonal antibodies, and analyzed by flow cytometry. Error bars depict standard deviations. (B) The protein level of CD55 and CD59 in A549 and Lovo cells were detected by western blotting. β-actin protein levels served as the loading control. CD55, decay accelerating factor; CD59, protectin.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD55 (cat. no. FHF055) and CD59 (cat. no. FHF059) mAbs were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

Techniques: Expressing, Incubation, Bioprocessing, Flow Cytometry, Western Blot, Control

Journal: Oncology Letters

Article Title: Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen

doi: 10.3892/ol.2018.8478

Figure Lengend Snippet: Establishing stable transfected α-gal-expressing cell lines. (A) α-1,3GT mRNA expression in A549, A549-V, A549-GT, Lovo, Lovo-V and Lovo-GT cells were detected by reverse transcription-polymerase chain reaction. The amplified product of α-1,3GT was detected by agarose gel electrophoresis in lane 2, 4 and 6 of the two gels. GAPDH was used as loading control in lane 1, 3 and 5 of the two gels. (B) Expression of α-gal epitope in each group of cells were detected by direct immunofluorescence (magnification, ×200). FITC-conjugated BS-IB4 lectin staining was performed to probe α-gal epitope. (B-a) A549-GT, (B-b) A549, (B-c) A549-V, (B-d) Lovo-GT, (B-e) Lovo, (B-f) Lovo-V and (B-g) positive control PIEC cells. (C) Expression of α-gal epitope in each group of cells were stained with FITC-BS-IB4 lectin, then analyzed by flow cytometry. Error bars depict standard deviations. FITC, fluorescein isothiocyanate; α-gal, Galα1-3Galβ1-4GlcNAc-R; α-1,3GT, α1,3-galactosyltransferase; PIEC, pig iliac arterial endothelial cells; A549-GT, α-gal expressing A549; A549-V, control.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD55 (cat. no. FHF055) and CD59 (cat. no. FHF059) mAbs were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

Techniques: Transfection, Expressing, Reverse Transcription, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Control, Immunofluorescence, Staining, Positive Control, Flow Cytometry

Journal: Oncology Letters

Article Title: Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen

doi: 10.3892/ol.2018.8478

Figure Lengend Snippet: Expression of CD55 and CD59 on α-gal-expressing cells influences their sensitivity to CDC. (A) A549, A549-V, A549-GT, Lovo, Lovo-V, Lovo-GT and positive control PIEC cells were incubated with various dilutions of NHS (0, 15, 30, 50%) and survival rates were analyzed by trypan blue staining. Error bars showed standard deviations (*P<0.05 vs. the control). (B) A549, A549-V, A549-GT Cells were pre-treated with various concentrations of PI-PLC (0.001, 0.01, 0.05, 0.1, 0.2, or 0.5 U/ml), incubated with 50% NHS, and survival rates were analyzed by trypan blue staining. Error bars showed standard deviations. *P<0.05, vs. the control. CD55, decay accelerating factor; CD59, protectin; NHS, normal human serum; PI-PLC, phosphatidylinositol-specific phospholipase C; A549-GT, α-gal expressing A549; A549-V, control.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD55 (cat. no. FHF055) and CD59 (cat. no. FHF059) mAbs were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

Techniques: Expressing, Positive Control, Incubation, Staining, Control

Journal: Oncology Letters

Article Title: Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen

doi: 10.3892/ol.2018.8478

Figure Lengend Snippet: Effects of PI-PLC treatment on CD55 and CD59 protein level in A549-GT cells. (A) Following 0.1 U/ml PI-PLC treatment, CD55 and CD59 were tested by western blot in A549, A549-V, A549-GT, Lovo, Lovo-V and Lovo-GT cells, compared with that prior to PI-PLC treatment. (B) After 0.1 U/ml PI-PLC treatment, A549-GT cells was incubated with fluorescein isothiocyanate-conjugated anti-human monoclonal antibodies. CD55 and CD59 were analyzed by flow cytometry, compared with that prior to PI-PLC treatment. Error bars showed standard deviations. *P<0.05 vs. the control. CD55, decay accelerating factor; CD59, protectin; PI-PLC, phosphatidylinositol-specific phospholipase C; A549-GT, α-gal expressing A549; A549-V, control.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD55 (cat. no. FHF055) and CD59 (cat. no. FHF059) mAbs were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

Techniques: Western Blot, Incubation, Bioprocessing, Flow Cytometry, Control, Expressing

Journal: Oncology Letters

Article Title: Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen

doi: 10.3892/ol.2018.8478

Figure Lengend Snippet: Effect of anti-CD55 and anti-CD59 on CDC in α-gal-expressing cells. The A549, A549-V and A549-GT cells were pre-incubated with each antibodies (anti-CD55, anti-CD59 and anti-CD55 with anti-CD59) (10 µg/ml), then 50% normal human serum was added and the survival rates were calculated. Error bars showed standard deviations. *P<0.05 vs. the control. CD55, decay accelerating factor; CD59, protectin; α-gal, Galα1-3Galβ1-4GlcNAc-R; A549-GT, α-gal expressing A549; A549-V, control.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD55 (cat. no. FHF055) and CD59 (cat. no. FHF059) mAbs were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

Techniques: Expressing, Incubation, Control

Journal:

Article Title: Reduced Sensitivity to Human Serum Inactivation of Enveloped Viruses Produced by Pig Cells Transgenic for Human CD55 or Deficient for the Galactosyl-?(1-3) Galactosyl Epitope

doi: 10.1128/JVI.78.11.5812-5819.2004

Figure Lengend Snippet: hCD55 and α-Gal expression on porcine cells. (a) Expression of CD55 was analyzed by FACs on both transgenic (TgPAE) and nontransgenic (PAE) pig cells. Staining by anti-hCD55 (shaded histogram), no primary antibody (negative control; solid line), or anti-hCD46 (dotted line, similar profile to that of the no-antibody control) followed by FITC-labeled secondary anti-mouse immunoglobulin antibodies (upper panels) is shown. Staining for α-Gal was a one-step incubation; hence, −lectin on the histogram represents cells only, and +lectin represents cells incubated with IB-4 lectin conjugated to FITC (lower panels). (b) α-Gal expression on ST-IOWA wild-type and ST-IOWA Gal-null (−/−) cells. Expression of the α-Gal antigen was analyzed by FACs, using the IB-4 lectin. A rightward shift of the histogram in the presence of lectin (+lectin) compared to the histogram generated in the absence of lectin (−lectin) indicates expression of the α-Gal antigen. The level of expression was determined using the MFI shift, generated by the Cell Quest FACScan software. The MFI shift was calculated by determining test MFI and subtracting that of negative controls (no primary antibodies for hCD55 and hCD46 expression and no lectin for α-Gal expression), and all the mean shifts are shown in Table ​Table11.

Article Snippet: Samples were then incubated on ice with either primary mouse anti-human CD55 antibody (BRIC110) or mouse anti-human CD46 antibody (J4-48) from Cymbus Bioscience Ltd., diluted to 1:20 in PBS/BA, for 1 h. After three washes in PBS/BA, samples were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch), diluted to 1:200 in PBS/BA and incubated on ice for 1 h. For α-Gal expression, cells were detached by a cell scraper, washed, resuspended, and stained in a single 1-h incubation with FITC-conjugated Bandeiraea simplicifolia isolectin (IB-4) (Sigma) at 10 μg/ml in PBS/BA on ice, as this lectin is specific for the terminal α-Gal sugar.

Techniques: Expressing, Transgenic Assay, Staining, Negative Control, Labeling, Incubation, Generated, Software

Journal:

Article Title: Reduced Sensitivity to Human Serum Inactivation of Enveloped Viruses Produced by Pig Cells Transgenic for Human CD55 or Deficient for the Galactosyl-?(1-3) Galactosyl Epitope

doi: 10.1128/JVI.78.11.5812-5819.2004

Figure Lengend Snippet: Summary of virus sensitivity and cell surface molecule expression

Article Snippet: Samples were then incubated on ice with either primary mouse anti-human CD55 antibody (BRIC110) or mouse anti-human CD46 antibody (J4-48) from Cymbus Bioscience Ltd., diluted to 1:20 in PBS/BA, for 1 h. After three washes in PBS/BA, samples were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch), diluted to 1:200 in PBS/BA and incubated on ice for 1 h. For α-Gal expression, cells were detached by a cell scraper, washed, resuspended, and stained in a single 1-h incubation with FITC-conjugated Bandeiraea simplicifolia isolectin (IB-4) (Sigma) at 10 μg/ml in PBS/BA on ice, as this lectin is specific for the terminal α-Gal sugar.

Techniques: Expressing

Journal:

Article Title: Reduced Sensitivity to Human Serum Inactivation of Enveloped Viruses Produced by Pig Cells Transgenic for Human CD55 or Deficient for the Galactosyl-?(1-3) Galactosyl Epitope

doi: 10.1128/JVI.78.11.5812-5819.2004

Figure Lengend Snippet: Demonstration of hCD55 incorporation on VSV particles by a viral pull-down assay. VSV harvested through HeLa, TgPAE A, and PAE E cells in the presence of antibody was incubated with protein G-expressing bacterial cells (OMNISORB). Antibodies used were three anti-human CD55 antibodies, BRIC 216, BRIC 471, and anti-DAF; anti-human CD46 J4-48 (hCD46); and anti-CD59 BRA-10G (hCD59). Five micrograms of anti-DAF and 1 μg of the other antibodies were used for incubation with virus and protein G cells. Titers of VSV for pulled-down and input particles were determined by TCID50 assay. The ratio of these titers is shown as percent binding.

Article Snippet: Samples were then incubated on ice with either primary mouse anti-human CD55 antibody (BRIC110) or mouse anti-human CD46 antibody (J4-48) from Cymbus Bioscience Ltd., diluted to 1:20 in PBS/BA, for 1 h. After three washes in PBS/BA, samples were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch), diluted to 1:200 in PBS/BA and incubated on ice for 1 h. For α-Gal expression, cells were detached by a cell scraper, washed, resuspended, and stained in a single 1-h incubation with FITC-conjugated Bandeiraea simplicifolia isolectin (IB-4) (Sigma) at 10 μg/ml in PBS/BA on ice, as this lectin is specific for the terminal α-Gal sugar.

Techniques: Pull Down Assay, Incubation, Expressing, TCID50 Assay, Binding Assay